1. Field of the Invention
The present invention relates generally to methods and compositions for expressing polypeptides in plant cell chloroplasts, and more specifically to chimeric constructs that allow for expression of a gene of interest in algae.
2. Background Information
Protein based therapeutics, or biologics, are the fastest growing sector of drug development, mainly due to the efficacy and specificity of these molecules. The specificity of biologics comes from their complexity, and biologics are only produced in living cells, making the production of these molecules time consuming and expensive. The expression of biologics in C. reinhardtii offers an attractive alternative to traditional mammalian-based expression systems, as the production of proteins in algae has inherently low costs of capitalization and production, and stable transgenic lines can be generated in a short period of time.
Recombinant technologies have allowed for the rapid identification of proteins capable of providing an array of therapeutic functions, and the utility of these types of molecules continues to grow as more sophisticated molecules and approaches are developed. As protein identification and engineering techniques have advanced, the need for more efficient and rapid production systems has emerged as a limiting factor in therapeutic protein production. A key consideration in the development of any new protein based drug is the inherent high cost of goods and capital investment associated with the production of these molecules.
Currently, there are a number of protein expression systems available for the production of biologics, and each system has different characteristics in terms of protein yield, ease of manipulation, and cost of goods. The majority of therapeutic proteins produced today come from the culture of transgenic Chinese Hamster Ovary (CHO) cells. Due to high capital and media costs, and the inherent complexity of CHO production systems, proteins produced in this manner are very expensive. E. coli is also used for biologic production, and bacterial expression is much faster and cheaper than CHO production, but bacteria are inefficient at producing properly folded complex proteins, and also show poor yields of many complex proteins.
Recently terrestrial plants have been used for biologic production. In plant systems, the therapeutic protein is synthesized within the plant cells and deposited into leaf or seed tissues. A variety of biologics have been produced in plants, including complex antibodies such as dimeric secretory immunoglobulin A molecules (sIgA). Protein production in plants is inherently less expensive than production by cell fermentation, but there are two major drawbacks to this approach. First, the length of time required from the initial transformation event to having usable (mg to gram) quantities of a protein can take up to two years for crops such as tobacco, and over three years for species such as corn. A second concern surrounding the expression of human therapeutics in crop plants is the potential for gene flow (via pollen) to surrounding food crops, and for the contamination of food supplies with transgenic seeds expressing human therapeutic proteins.
Compared to land plants, algae like C. reinhardtii grow much faster, doubling in approximately 8 hours. As C. reinhardtii propagates by cell division, the time from initial transformation to protein production is significantly reduced relative to higher plants or mammalian cells, requiring as little as three weeks to generate stable transgenic lines, with the potential to scale up to production volumes in another four to six weeks. Algae propagate by vegetative replication, lack pollen, and have no potential for gene transfer to food crops. C. reinhardtii can easily be grown in containment, again reducing any chance of environmental contamination. Growth in containment also assures that external contaminants, like pesticides or pollutants, do not contaminate the protein being produced. Algae are eukaryotes, meaning that unlike bacteria, they are efficient at producing complex proteins and have the machinery necessary to fold and assemble multi-component complexes into functional proteins. In addition, green algae are generally regarded as safe (GRAS), posing little risk of viral, prion or bacterial endotoxin contamination. Thus, algae would seem to be an ideal system for biologic production, as long as high levels of protein expression can be achieved, and that the expressed protein can be shown to function in a bioactive manner.